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Aim: Depression is highly prevalent in type 2 diabetes and is associated with lower adherence to medical treatments, worse glycemic control, and increased risk for diabetes-related complications. The mechanisms underlying depression in type 2 diabetes are unclear. The haptoglobin (Hp) genotype is associated with type 2 diabetes related complications including increased risk for cerebrovascular pathology and worse cognitive performance. Its relationship with depression is unknown. We investigated the role of Hp genotype on the association of depression with brain and white matter hyperintensities (WMH) volumes. Methods: Depressive symptoms (measured with the 15-item Geriatric Depression Scale), brain MRI, and Hp genotypes, were examined in elderly subjects with type 2 diabetes [29 (13.8%) Hp 1-1 carriers and 181 (86.2%) non-carriers]. The interaction of Hp genotype with number of depressive symptoms on regional brain measures was assessed using regression analyses. Results: The significant interactions were such that in Hp 1-1 carriers but not in non-carriers, number of depressive symptoms was associated with overall frontal cortex (p = 0.01) and WMH (p = 0.04) volumes but not with middle temporal gyrus volume (p = 0.43). Conclusions: These results suggest that subjects with type 2 diabetes carrying the Hp 1-1 genotype may have higher susceptibility to depression in the context of white matter damage and frontal lobe atrophy. The mechanisms underlying depression in diabetes may differ by Hp genotype.

The aims of this study were to assess the feasibility of a randomised controlled trial of foot care using patients recruited from the retinal imaging clinic, and to determine whether in addition to standard care and advice, giving patients with diabetes a VibraTip would enhance confidence in foot self-care. Sixty subjects recruited in retinal imaging clinics were randomised to receive a standard foot care leaflet, or leaflet plus VibraTip with a demonstration of its use. VibraTip recipients were encouraged to take it home but given no further instructions. Participants were asked to complete a ‘confidence in self-care’ questionnaire at enrolment and follow up, six months later. Recruitment was completed in three, all-day clinics. Follow-up rates were also high with only one subject lost from each group. Confidence in the ability to check feet for sores or blisters rose in the control group from 90.4% to 96.6% and fell from 90% to 88.6% in the VibraTip group. The feasibility results indicate that large randomised controlled trials in this patient group would be achievable for short educational interventions. Although only a feasibility trial, the study suggested that, if anything, providing VibraTip might actually cast doubt in the minds of patients with diabetes who had been very confident that they could look after their feet. Self-confidence may be more likely to foster complacency than behaviours likely to minimise risk of harm.

Rapid but often transient changes in mitotic and apoptotic activity are important components of the pituitary response to changes in the hormonal environment. For example, bilateral adrenalectomy and orchidectomy each result in a wave of increased mitosis lasting approximately 1 week, mediated by the same population of trophically active and, to a large extent, endocrinologically inactive cells. By contrast to these tonic inhibitors of pituitary trophic activity, reports of a progressive increase in lactotroph numbers during pregnancy suggest that oestrogen is a potent and persistent pituitary mitogen. By comparing the amplitude and duration of male rat anterior pituitary mitotic responses to oestrogen treatment, to adrenalectomy, and to a combination of the two, the present study aimed to further clarify the characteristics of the oestrogen-induced trophic response, in particular whether lactotrophs are the predominant cell type involved. Adrenalectomy produced a wave of increased mitotic activity, which resolved within 7 days as expected, whereas oestrogen induced a significant increase in mitotic activity, which was sustained for the 14-day duration of the study. The trophic effects of combining adrenalectomy and oestrogen treatment were not additive in that the statistically insignificant upward trend in mitotic index during the first few days compared to oestrogen treatment alone was entirely abolished by oestrogen pre-treatment. The increase in mitotic activity in lactotrophs induced by oestrogen either with or without adrenalectomy did not result in an increase in the relative size of the prolactin-positive compared to prolactin-negative pituitary parenchymal cell numbers by the end of the study. Despite the marked increase in the lactotroph population that is reported during pregnancy, these data indicate that at least the early (i.e. within 2 weeks) mitotic response to pharmacological doses of oestrogen increases mitotic activity in the lactotroph subpopulation by only 5-8% relative to other cellular subpopulations. Unexpectedly, the mitotic response to oestrogen principally occurs in non-prolactin-containing cells and results in the recruitment, amongst other trophically responsive populations, of the entire subpopulation of prolactin-, adrenocorticotrophic hormone- and luteinising hormone-negative cells that respond mitotically to adrenalectomy. Oestrogen therefore has a previously unrecognised non-cell type-specific trophic effect in the pituitary that obscures the relative expansion of the lactotroph population by inducing concurrent increases in numbers of prolactin-negative cells, the nature of which at least in part remains to be determined.

Oestrogen is a powerful mitogen that is believed to exert a continuous, dose-dependent trophic stimulus at the anterior pituitary. This persistent mitotic effect contrasts with corticosterone and testosterone, changes in the levels of which induce only transient, self-limiting fluctuations in pituitary mitotic activity. To further define the putative long-term trophic effects of oestrogen, we have accurately analysed the effects of 7 and 28 days oestrogen treatment on anterior pituitary mitotic activity in ovariectomized 10-week-old Wistar rats using both bromodeoxyuridine (BrdU) and timed colchicine-induced mitotic arrest. An oestrogen dose-dependent increase in mitotic index was seen 7 days after the start of treatment as expected, representing an acceleration in gross mitotic activity from 1.7%/day in ovariectomized animals in the absence of any oestrogen replacement to 3.7%/day in the presence of a pharmacological dose of oestrogen (50 mcg/rat per day: ∼230 mcg/kg per day). Despite continued exposure to high-dose oestrogen and persistence of the increase in pituitary wet weight, the increase in mitotic index was unexpectedly not sustained. After 28 days of high-dose oestrogen treatment, anterior pituitary mitotic index and BrdU-labelling index were not significantly different from baseline. Although a powerful pituitary mitogen in the short term, responsible, presumably, for increased trophic variability in oestrus cycling females, these data indicate that in keeping with other trophic stimuli to the pituitary and in contrast to a much established dogma, the mitotic response to longer-term high-dose oestrogen exposure is transient and is not the driver of persistent pituitary growth, at least in female Wistar rats.

A significant proportion of pituitary macroadenomas, and by definition all microadenomas, regain trophic stability after an initial period of deregulated growth. Classical proto-oncogene activation and tumor suppressor mutation are rarely responsible, and no histologic or molecular markers reliably predict behavior. GNAS1 activation and the mutations associated with multiple endocrine neoplasia type 1 and Carney complex, aryl hydrocarbon receptor interacting protein gene mutations, and a narrowing region of chromosome 11q13 in familial isolated acromegaly together account for such a small proportion of pituitary adenomas that the pituitary adenoma pathogenic epiphany is surely yet to come.

The male rat anterior pituitary responds highly reproducibly to specific hormonal stimuli in terms of the extent and timing of mitotic and apoptotic (trophic) activity. The principal objective of the present study was to define the contribution of hormonally identifiable cells to the trophic responses to bilateral gonadectomy and bilateral adrenalectomy. The patterns of pituitary mitotic responses to adrenalectomy and gonadectomy are similar in amplitude and duration. When adrenalectomy and gonadectomy are combined, the amplitude of the pituitary mitotic response is unchanged. That is, the trophic stimuli are not additive. Dexamethasone-induced apoptosis in nascent cells is amplified not only by recent adrenalectomy, but also, and to an almost identical extent, by gonadectomy. Combining adrenalectomy and gonadectomy does not further enhance the size of the apoptotically-responsive cell population. Dual bromodeoxyuridine and adrenocorticotrophic hormone (ACTH) or luteinising hormone (LH) immunolabelling showed that more than 95% of all dividing cells are not and do not become positive for either of these hormones during the period of peak mitotic response. Following adrenalectomy, most newly-formed ACTH cells are derived from differentiation of pre-existing hormonally undifferentiated cells. Despite an overall increase in mitotic activity, there is no measurable increase in the number of LH immunopositive cells after gonadectomy. The nonadditive pituitary mitotic and apoptotic responses to adrenalectomy and gonadectomy strongly suggest that the same progenitor cell population responds mitotically to both. This weakens the prevailing view that hormonally identifiable cells with specific trophic profiles contribute significantly to pituitary cell subpopulation revision.

We have used a direct, non-immunochemical and highly accurate method to quantify the effects of testosterone and oestrogen on mitotic and apoptotic activity in the young, male rat anterior pituitary in vivo. Surgical gonadectomy resulted in a 3-fold increase in mitotic activity by the fourth post-operative day, which returned gradually to levels seen in intact animals over the subsequent 3–4 weeks. Both a single dose of Sustanon, a mixture of long-acting testosterone esters in arachis oil, and the same dose divided over 7 days (starting 6 days after gonadectomy), initially suppressed mitotic activity to levels seen in intact animals, but was associated after 48–96 h with a wave of increased mitotic activity. The latter was blocked by co-administration of Sustanon with the non-steroidal aromatase inhibitor letrozole and was not seen when the non-aromatisable androgen dihydrotestosterone was substituted for Sustanon. Oestrogen alone in gonadectomised and intact rats produced a marked increase in mitosis as expected. With the exception of a transient increase in response to a single high-dose injection of Sustanon in gonadectomised animals, apoptotic activity was unaffected by all of the above. This study suggests that pituitary mitotic activity is tonically inhibited by gonadal hormone production (at least in the short term) in adult male rats. The study also suggests that supraphysiological testosterone treatment – while unable to reduce anterior pituitary mitotic activity in untreated, intact animals –suppresses the early increase in mitotic activity induced by gonadectomy. Oestrogen, either exogenous or generated locally by aromatisation, stimulates anterior pituitary mitotic activity in a time-dependent manner.

We have previously identified a series of age-dependent, temporally constrained and closely interdependent mitotic and apoptotic events in the male rat anterior pituitary that occur in response to timed single and repeated hypothalamo-pituitary-adrenal axis stimuli. One of the most dramatic of these is the short burst of apoptosis that occurs 24-48 h after exposure to dexamethasone. If bilateral adrenalectomy precedes exposure to dexamethasone by 1-2 weeks, mitotic activity is transiently increased and the subsequent apoptotic response to dexamethasone greatly enhanced. This study was designed to determine whether adrenalectomy-induced augmentation of the apoptotically sensitive pituitary cell population is mediated via glucocorticoid withdrawal at the level of the pituitary, or whether increased exposure to hypothalamo-hypophyseal trophic hormones of paraventricular origin is responsible. We used stereotaxic surgery to isolate both paraventricular nuclei without disturbing either median eminence input from the arcuate and supraoptic nuclei, or the hypothalamo-hypophyseal-portal blood flow that carries a significant proportion of the pituitary systemic supply. When bilateral adrenalectomy and paraventricular nucleus disconnection were combined, the adrenalectomy-induced increase in anterior pituitary pro-opiomelanocortin (POMC) transcript prevalence was abolished, confirming the loss of paraventricular corticotrophin-releasing hormone (CRH) input. However, the amplitude and pattern of the adrenalectomy-induced anterior pituitary mitotic response and enhancement of the apoptotic response to dexamethasone 1 week later remained completely intact. These data demonstrate that anterior pituitary trophic responses following bilateral adrenalectomy are more likely to be mediated through direct glucocorticoid withdrawal at the level of the pituitary rather than via changes in hypothalamo-hypophyseal releasing factor exposure. This finding highlights the presence of distinct control systems for pituitary hormone gene expression and pituitary mitotic and apoptotic responses.

The anterior pituitary is active mitotically and apoptotically under basal conditions and in response to a variety of physiological and pathophysiological stimuli. Hypothyroidism in man is associated with a modest but very occasionally dramatic increase in overall pituitary size. The mechanisms underlying this reversible phenomenon remain obscure. In the present study we have examined young adult rat anterior pituitary following surgical thyroidectomy and subsequent thyroid hormone treatment and withdrawal using an extremely accurate system for quantifying directly identified mitotic and apoptotic events. Despite the expected increase in the number and/or proportion of immunohistochemically identifiable thyrotrophs three weeks after thyroidectomy, mitotic and apoptotic activity remained unchanged, as did pituitary wet weight, in comparison with sham-operated and intact controls. In contrast, mitotic but not apoptotic activity was enhanced by treatment of thyroidectomized animals with thyroid hormones (triiodothyronine (T3) and thyroxine (T4) 1.8 microg and 3.6 microg/100 g body weight per day respectively), and once again declined to levels seen in intact animals within 72 h of subsequent thyroid hormone withdrawal. Thyroid hormone-induced enhancement of mitotic activity was also seen in intact rats treated with similar doses of thyroid hormones for 7 days and in thyroidectomized rats treated for a similar period with very low dose thyroid hormone replacement at a level that had no effect on raised hypothalamic TRH- or pituitary TSHbeta-transcript prevalence (0.018 microg T3 plus 0.036 microg T4/100 g body weight per day). Thus changes in mitotic and apoptotic activity are unlikely to be the principle mechanism for the apparent increase in thyrotrophs up to 4 weeks after thyroidectomy. In contrast, the data indicate that thyroid hormones have a permissive effect on anterior pituitary mitotic activity in thyroidectomized male rats. Thyroid hormone-induced enhancement of mitotic activity in intact rats further suggests that in euthyroid rats, ambient thyroid hormone levels are a limiting factor for anterior pituitary mitotic activity. In summary, this time course study of young, male rats has shown for the first time that thyroidectomy, thyroid hormone replacement and subsequent withdrawal has no significant effect on anterior pituitary apoptotic activity. Secondly, it has shown that the anterior pituitary mitotic response to thyroidectomy is blocked by complete thyroid hormone deprivation, but can be restored by very low level thyroid hormone replacement, and thirdly that in intact animals thyroid hormone levels significantly limit anterior pituitary mitotic activity.

Secretion of adrenocorticotropic hormone (ACTH) from the fish pituitary, which occurs in times of stress, is stimulated by several hypothalamic neuropeptides, one of which is arginine vasotocin (AVT). This study investigates whether gene expression for AVT is up-regulated during acute or chronic stress. Rainbow trout (Oncorhynchus mykiss) were subjected to one of two forms of acute stress-either 2 h confinement followed by 2 h recovery, or capture and transfer to low water for 2 min followed by 4 h recovery in their home tank before autopsy. In other experiments, these stresses were repeated daily for 5 or 6 days (chronic stress). Quantification of AVT transcript prevalence in the parvocellular and magnocellular neurones of the preoptic nucleus after in situ hybridization was used as a monitor of the AVT gene response to stress. The results showed that acute confinement, but apparently not brief low-water stress, significantly increased AVT transcript prevalence in a group of parvocellular perikarya. When applied repeatedly, both forms of stress caused habituation, such that the AVT hybridization signal remained at control or even lower levels despite elevated pro-opiomelanocortin transcripts in the corticotropes and raised plasma cortisol concentrations. The AVT hybridization signal in the magnocellular perikarya showed no significant response to either acute or chronic stress. The results support the idea that these parvocellular AVT neurones are involved in ACTH stimulation during acute stress, and that the system habituates to chronic stresses.

Many millions of people throughout the world are at risk of developing iodine deficiency-associated disorders. The underlying effects of iodine deficiency on neuroendocrine function are poorly defined. We have studied stress-induced and diurnal variation in corticosterone secretion in female rats rendered chronically hypothyroid by feeding them an iodine-free diet for 6 months. Corticosterone secretory responses in iodine deficient animals were compared to those seen in animals rendered hypothyroid with propylthiouracil and untreated controls. By using a well-validated, automated blood sampling system to collect small samples of blood over the complete daily cycle in unrestrained animals, we have demonstrated for the first time that the normal diurnal rhythm of corticosterone secretion is lost in chronic iodine deficiency and that the corticosterone secretory response to the psychological stress of 10 min exposure to white noise is attenuated. Despite restoration of circulating triiodothyronine and thyrotropin releasing hormone- and thyroid stimulating hormone beta-transcript prevalence in the hypothalamus and pituitary, respectively, 1 month after restoration of normal iodine-containing diet both the diurnal variation in corticosterone levels and the corticosterone secretory response to the noise stress remained reduced in amplitude compared to control animals. Thus, chronic hypothyroidism induced by iodine deficiency significantly attenuates hypothalamo-pituitary-adrenal axis activity, an effect that persists after functional recovery of the thyroid axis.

To further define the neuroendocrine consequences of intrauterine growth retardation (IUGR), we have used a rat model of maternal protein restriction throughout pregnancy to examine the pattern of corticosterone and GH secretion under basal conditions and in response to psychological stress in male offspring at 4, 9, and 18 months of age. The findings were correlated with studies of behavioral activity. Despite a consistent reduction in birth weight and failure of catch-up growth, there were no significant differences in GH secretory profiles between IUGR and control rats at any age. We were unable to demonstrate a difference in the number, amplitude, length, or area of corticosterone secretory pulses between control and IUGR animals; although again, there was a significant decrease with age. The mean peak plasma concentration of corticosterone in response to a noise stress also declined with age but was unaffected by IUGR. There were no consistent, statistically significant differences in behavioral responses between normal control and IUGR animals or between groups of animals at different ages. These results do not, therefore, support the presence of major functional abnormalities in either GH or corticosterone secretory responses in adult male rats subjected to IUGR.

Previous studies on trout suggest that melanin-concentrating hormone (MCH) acts at both hypothalamic and pituitary levels to restrain the release of adrenocorticotropic hormone and hence cortisol during stress. Using in situ hybridization, the present work examined whether high rates of MCH secretion were associated with changes in the synthesis of arginine vasotocin (AVT), one of the corticotropin secretogogues. It also examined whether high endogenous MCH secretion restrains cortisol secretion during intense as well as mild stress, and how exogenous MCH affects the rise in plasma cortisol following injection stress. Trout were reared in black- or white-coloured tanks for 1 year or more to achieve maximal differences in MCH secretion. Following a mild stress, cortisol secretion was greater in black-reared fish with low MCH secretion which is in line with previous findings but, following a more severe stress, plasma cortisol concentrations were similar in the two groups. Injection of MCH into black-adapted fish restrained the stress-induced rise in plasma cortisol concentration during the first hour but did not affect final cortisol values. In two separate experiments, AVT mRNA levels were significantly lower in the hypothalamus of black-reared fish. Possible explanations for this include a greater negative-feedback restraint by cortisol, which is likely to rise higher in black-adapted fish during the moderate, daily stresses of aquarium life; or the possibility that exposure to a white background may be psychologically stressful, stimulating AVT transcription. The possibility that MCH directly stimulates AVT transcription cannot be excluded but seems less likely. The results suggest that while MCH may restrain the release of hypothalamo-pituitary stress hormones under moderately stressful conditions, it does not restrain AVT synthesis.

Glucocorticoid withdrawal, depending on the dose and duration of treatment, results in a transient but sometimes prolonged reduction in hypothalamo-pituitary-adrenal (HPA) axis secretory responsiveness. As the anatomic basis of HPA axis suppression remains uncertain, we have directly examined changes in trophic activity within the rat anterior pituitary gland following dexamethasone withdrawal and re-treatment. Treatment of adrenalectomised, male Wistar rats with dexamethasone results in a discrete, highly significant burst of apoptosis in the anterior pituitary with concurrent suppression of mitosis. Despite a surge in mitotic activity immediately after dexamethasone withdrawal, calculated total anterior pituitary cell populations remain below that seen in untreated adrenalectomised controls. Repeated exposures to dexamethasone show that the dexamethasone-sensitive cell population that is deleted by apoptosis is partially but not completely restored. As the amplitude of apoptotic bursts induced by second and third dexamethasone exposures are similar but smaller than that induced by initial exposure, it appears that the very first exposure to dexamethasone deletes a subset of anterior pituitary cells that are either not restored at all, or are only replaced very slowly. The reduced proportion of corticotrophs contributing to the increase in mitotic index after dexamethasone withdrawal corroborates this. Although continued cell turnover within the pituitary predicts that the absolute cellular deficit would diminish with time, the effects seen may contribute to the delayed recovery of pituitary axis function following cessation of glucocorticoid treatment.

Testing vibration sense is a recognised component of neuropathy assessment. One hundred consecutive patients attending diabetic annual review and podiatry clinics were enrolled in a study to compare a 10 g Semmes–Weinstein monofilament and a 128Hz tun ing fork with VibraTip ® , a novel, key-fob-sized source of fixed amplitude vibration for the identification of peripheral neuropathy. Patients were tested sequentially with the three devices in random order on either the left or right foot. Of the 100 patients 55 felt both the vibrating tuning fork and VibraTip, the remaining 45 patients felt neither. Pressure from the 10 g monofilament was perceived by 53 patients of whom 51 also felt VibraTip ® . VibraTip ® proved a practical, hygienic, simple, rapid and very specific test of the integrity of vibration sense, easily controlled with a null stimulus, that appeared highly engaging for patients. As the utility of testing for neuropathy is no more or less than a strategy to persuade patients to change their behaviour, VibraTip ® may be a useful addition to sensory nerve function testing at the clinical interface. Br J Diabetes Vasc Dis 2010;10:284-286.

Summary The objective of clonality analysis is to help differentiate hyperplasia from neoplasia. If the clonality of different pituitary subpopulations is skewed rather than uniform it is possible that a physiological trophic response could, by displacing trophically inert cells, give rise to a potentially evanescent monoclonal expansion in the absence of spontaneous somatic mutation. Such a clonal temtory model would allow for the presence of cellular subsets (a feature difficult to explain in terms of classical neoplasia) and account for a number of unusual characteristics of pituitary adenomas including monoclonality, cyclical activity, lack of oncogene involvement. spontaneous resolution and quantitative but not qualitative changes in secretory activity seen, for example, in Cushing's disease.

Erectile dysfunction is a common and distressing medical condition that is now highly amenable to treatment almost irrespective of the cause. Safe, non-surgical treatments with unequivocal efficacy are psychological therapy, intracorporeal injection of vasoactive drugs, transurethral vasodilators and oral sildenafil, all of which have been reported to have a 50-70% overall response rate. Vacuum constriction devices are acceptable for some, usually older patients and oral yohimbine is thought to have marginal efficacy. Local creams to induce or enhance erectile function are currently being investigated. There is no place for androgen supplementation unless the patient is profoundly hypogonadal. Treatment of hyperprolactinaemia is very effective but is a rare cause of erectile dysfunction. As intercourse may entail an unfamiliar level of physical activity, it is sensible to ensure that the patient is able to climb a flight or two of stairs comfortably without provoking undue breathlessness or chest pain and to provide suitable advice about technique before commencing treatment. Once it is clear to the patients that erectile dysfunction can be satisfactorily overcome, the long-term use of treatments to do so tends to wane. Thus, although the prospect of effective treatment for what had been for many a distressing life sentence has the potential to place new demands on the health service, there is no evidence that restrictions on prescribing will prove economically rational.

Direct analyses of trophic activity in the pituitary have been hampered by the lack of normal human tissue to study and by the short duration of the histologically overt phases of mitosis and apoptosis, which renders significant trophic events difficult to quantify. In rats and dogs, pituitary cell turnover is rapid in youth, declines markedly with age and virtually ceases by ‘late middle age’. Specific stimuli superimpose brief but dramatic trophic events on this active background. There is little convincing evidence, as yet, for plasticity, i.e. the persistence of cell population changes after transient stimuli have passed. In contrast to spontaneous pituitary adenomas in rats, human pituitary tumours show relatively modest increases in mitotic activity. In the light of these observations, this chapter examines the accepted models of pituitary adenoma formation and propagation, with special reference to trophic activity, clonality and tumour behaviour.

OBJECTIVE Nitric oxide (NO) has been implicated in the control of the secretory response to growth hormone-releasing hormone (GHRH) and may also modify GH release in response to excitatory aminoacids. Although rat and mouse pituitary cell lines have been shown to express neuronal NO synthase (nNOS), there has until now been no information on nNOS gene expression in human pituitary adenomas. Our objective was to provide such data and correlate the presence of nNOS transcripts with GHRH transcripts. PATIENTS Pituitary adenoma tissue was obtained from a random selection of 32 patients with somatotrophadenomas, 16 patients with corticotroph adenomas, 39 patients with endocrinologically inactive adenomas and nine patients with macroprolactinomas undergoing transsphenoidal hypophysectomy. MEASUREMENTS Transcripts for nNOS and GHRH were indentified in frozen tissue sections by in situ hybridization histochemistry using synthetic 35 S-labelled oligodeoxynucleotide probes with 100% homology to the target transcript. RESULTS Neuronal NOS transcripts were identified in one of 16 corticotroph adenomas (6%), one of nine macroprolactinomas (11%), six of 39 endocrinologically inactive adenomas (15%) and 13 of 32 somatotroph adenomas (41%). GHRH transcripts were found in a similar distribution to nNOS transcripts in 10 of the 13 nNOS-expressing somatotroph adenomas, and in three of the four remaining adenomas from which suitable tissue was available. Cross-hybridization of the nNOS and GHRH probes to the same target was excluded by including rat brain sections cut through the arcuate nucleus as hybridization controls. Furthermore, two different nNOS oligodeoxynucleotide probes complementary to different regions of the target transcript produced identical results. CONCLUSIONS These results suggest that there is a close correlation between nNOS gene expression and 'ectopic' expression of GHRH in human pituitary tumours, especially somatotroph adenomas. The relevance of these findings from a functional or pathologenic viewpoint remains unclear, but the data again emphasize that it is not just GH secretion that distinguishes somatotroph adenomas from other pituitary tumours.

We have examined the effects of isotonic hypovolemia on the expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat, using in situ hybridization histochemistry with a 35S-labelled oligodeoxynucleotide probe complementary to nNOS mRNA. Intraperitoneal (i.p.) administration of polyethylene glycol (PEG) (MW 4000, 20 ml/kg body weight) dissolved in 0.9% saline (20% w/v) induced isotonic hypovolemia. The expression of the nNOS gene in the PVN and SON 6 h after i.p. administration of PEG was increased significantly in comparison with controls. The dual staining for NADPH diaphorase activity and Fos-like immunoreactivity (Fos-LI) showed that at 3 and 6 h after i.p. administration of PEG, a subpopulation of NADPH diaphorase-positive cells in the PVN and SON exhibited nuclear Fos-LI. These results suggest that NO in the PVN and SON may be involved in the neuroendocrine and autonomic responses to non-osmotic hypovolemia.

We have used an extremely accurate, dedicated, real time computerized image analysis system to facilitate the manual quantification of changes in the prevalence of mitotic figures and apoptotic bodies in male rat pituitary following surgical adrenalectomy and, 14 days later, dexamethasone treatment. Under basal conditions, the prevalence of mitotic figures and apoptotic bodies was 0.066+/-0.016% and 0.030+/-0.012% (mean+/-SE) respectively. Dexamethasone treatment reduced the prevalence of mitotic figures and, in adrenalectomized animals, produced a highly significant and reproducible burst of apoptotic activity that peaked 48 h after the beginning of treatment (0.261+/-0.022%) before falling sharply to control levels within a further 8 h. Two weeks after the start of dexamethasone treatment, total pituitary cell numbers continued to decline. The rate of accumulation of mitotic figures in vivo after colchicine treatment indicates that mitosis is histologically overt in 2 microm thick hematoxylin and eosin stained sections under the light microscope for around 80 min; that apoptosis--identified as classical apoptotic bodies--is overt for 44 min and that, on average, a young, adult, male rat anterior pituitary cell either dies or divides as frequently as once every 60-70 days. These data show that transient and apparently trivial fluctuations in the prevalence of apoptotic and mitotic events have a profound effect on pituitary cell population dynamics, and demonstrate that dexamethasone treatment of adrenalectomized rats produces a decline in total anterior pituitary cell numbers that continues for at least 2 weeks after the start of glucocorticoid treatment.

Several earlier studies have shown that some human pituitary GH-secreting somatotrophinomas are able to synthesise and release hypothalamic GHRH and it has been proposed that a positive autocrine feedback loop involving this tumor-derived GHRH may participate in tumorigenesis. We have used in-vitro cell culture and exploited an antagonist to GHRH, (Ac-Tyr1,D-Arg2)-GHRH (1-29)-amide (GHRH-A), to further investigate whether an autocrine loop involving somatotrophinoma-derived GHRH may exist. In situ hybridization demonstrated presence of GHRH transcripts in 5 of 9 human somatotrophinomas. In culture, GHRH-A failed to inhibit basal release of GH or production of cAMP irrespective of presence or absence of GHRH transcripts. However, GHRH-A was able to completely or partially abolish the stimulatory effects of exogenously added GHRH peptide. Additionally, the average stimulatory effect of exogenous GHRH on in vitro GH secretion by somatotrophinomas possessing GHRH mRNA was identical to that shown by tumors not expressing the GHRH gene. Whilst confirming that many human pituitary somatotrophinomas are able to express the GHRH gene, the failure of GHRH-A to inhibit basal GH secretion argues against the concept of the existence of an autocrine stimulatory loop involving secreted GHRH peptide.

Corticotropin-releasing hormone and arginine vasopressin gene transcription were studied in the parvocellular cells of the hypothalamic paraventricular nucleus in response to both adrenalectomy and the injection of corticosterone. Following 6 days ADX, there was an increase in AVP mRNA, AVP hnRNA and CRH mRNA but not CRH hnRNA. After injection of corticosterone in ADX rats there was an extremely rapid reduction in AVP hnRNA which fell markedly within 15 min. AVP mRNA, however, remained raised throughout the 4 h. CRH hnRNA fell at 2 and 4 h, while CRH mRNA was only significantly reduced at the 4 h time point. Data demonstrate markedly different kinetics for the glucocorticoid regulation of the AVP and CRH genes in the hypothalamus.

The hypothalamic-pituitary-adrenal axis adapts to periods of chronic or repeated stress. We have now investigated whether there is any differential effect of repeated immobilization stress on CRH and arginine vasopressin (AVP) gene transcription in the hypothalamic paraventricular nucleus (PVN), using probes for both intronic and exonic sequences of the AVP and CRH genes. Daily restraint for 13 days had no significant affect on basal corticosterone level, CRH heteronuclear RNA (hnRNA), CRH messenger RNA (mRNA), AVP hnRNA, or AVP mRNA. After the final episode of acute restraint, on day 14, there was a rapid increase in plasma corticosterone and AVP hnRNA level in the parvocellular subdivision of the PVN, a slower increase in AVP mRNA level in the parvocellular subdivision of the PVN, but no significant change in either CRH hnRNA or CRH mRNA in the PVN. The increase in AVP hnRNA and mRNA was specific to the parvocellular PVN, because there was no change in either transcript in the magnocellular cells of thi...

This paper describes a method for infusing chronically substances into the cranial cavity of free-living rainbow trout Oncorhynchus mykiss for several weeks. The efficacy of the method was established by examining the penetration of radioactively labelled phosphorothioate oligodeoxynucleotides and a blue-coloured dye, xylene cyanole, into brain tissue. No problems with pump patency were encountered, and the contents of the pump diffused consistently throughout the brain ventricular system, including the anterior lateral ventricles of the olfactory lobes, the third ventricle under the optic tecta and into the hypothalamus, including the lateral ventricular recesses. Autoradiographic examination of frozen sections demonstrated variable penetration of labelled probe into brain interstitium to a depth of up to approximately 200 μm. At the end of the experiment, >50% of radioactivity within brain tissue was shown to be of similar size to intact, labelled oligodeoxynucleotides.

We have used in situ hybridization histochemistry to investigate the effects of maternal thyroidectomy and chronic maternal iodine deficiency on basal neuroendocrine function in rat pups. Specifically, we have measured hypothalamic thyrotrophin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) expression together with circulating levels of tri-iodothyronine (T3) in rat pups delivered from and suckled by thyroidectomized or iodine-deficient dams. Because of the close interaction between the thyroid, adrenal and growth hormone axes, we have also examined hypothalamic corticotrophin-releasing hormone (CRH) and growth hormone-releasing hormone (GRH) transcripts at the same time points: birth, 1 month and 2 months of age. Three weeks after surgical thyroidectomy, adult female Sprague–Dawley rats proved unable to carry pups to term and lactate successfully. Pups delivered from thyroidectomized dams given a small replacement dose of T3 during pregnancy were significantly lighter than controls (84 ± 3%) and had markedly depressed plasma T3 levels (36 ±6% of control). Hypothalamic CRH and GRH transcript levels were significantly decreased in pups at birth (to 8±2·5% and 24 ± 8% of control respectively) but had returned to normal by 1 month after delivery. Pituitary TSH transcript levels and hypothalamic levels of TRH transcripts, however, were similar to those of controls. Only one of seven dams fed a low-iodine diet for 6 months produced live pups, and these were too few in number to produce significant data. Dams fed a lowiodine diet from 4 months before mating, however, did produce live pups and although they were not significantly lighter than control pups at birth, by 1 month after birth, they were significantly lighter (72 ± 3% of controls). Circulating T3 levels were not significantly different from control at any time point examined. Hypothalamic TRH levels were significantly elevated at birth (451 ± 138% of control), but this difference was not maintained at 1 or 2 months after birth despite the lactating dams being maintained on the low-iodine diet. Pituitary TSH levels showed an upward trend at all time points that reached significance at 1 month after birth (204 ± 19%; P In summary, chronic iodine deficiency or thyroidectomy with low-level T3 replacement in Sprague–Dawley rats markedly impaired fertility and the ability to carry pups to term, and produced an unexpectedly modest up-regulation of the hypothalamo–pituitary–thyroid axis and down-regulation of the hypothalamo–pituitary–adrenal axis. Journal of Endocrinology (1997) 152, 423–430

The rapid detection of gene activation is important for our understanding of gene regulation. We have therefore studied heteronuclear (i.e. nascent) RNA (hnRNA) by using 35S-labelled corticotrophin-releasing hormone (CRH) riboprobes and arginine vasopressin (AVP) oligonucleotide probes directed against intronic and exonic sequences of both CRH and AVP transcripts for in situ hybridization studies of transcriptional changes during acute stress. CRH and AVP intronic signals (found in newly synthesized transcripts) were confined to the nuclei of the parvocellular cells in the paraventricular nucleus (PVN) whilst CRH and AVP exonic signals (found in both newly formed and mature transcripts) were primarily located in the cytoplasm of these cells. AVP hnRNA and mRNA were also present at high levels in the magnocellular PVN. The levels of CRH hnRNA and parvocellular AVP hnRNA in the PVN were significantly increased 1 and 2 h after the onset of restraint. The levels of CRH mRNA on the other hand were not significantly increased until 4 h after the onset of restraint. The number of AVP mRNA-expressing neurons in the medial parvocellular cells of the PVN significantly increased at 2 h and peaked at 4 h after the onset of stress. In contrast, densitometric analysis indicated that the increase in AVP mRNA levels in these cells did not reach significant difference from control until 4 h after the onset of restraint. There were no significant changes in AVP hnRNA or AVP mRNA levels in the magnocellular subdivision of PVN at any time point after the onset of restraint. Journal of Endocrinology (1997) 152, 81–89

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

Pituitary carcinoma with metastatic endocrine secreting tissue is rare. Eight cases of malignant, growth hormone-secreting tumours, all metastatic within the central nervous system have been previously described. Complete surgical resection was not possible and the patients died within 4 years of presentation with distant spread. Here we describe the first case of an apparent surgical cure of a somatotroph carcinoma metastatic to the cervical spine, documented by biochemical assessment and magnetic resonance and radioligand imaging. The possibility that intrathecal metastasis of somatotroph tumours may be responsible for some cases of treatment resistant acromegaly is discussed.

We have recently identified gonadotropes as target cells for ATP action via ATP receptors of the P2U subtype. The present studies have used gonadotrope-derived alpha T3-1 cells to examine the possible signaling mechanisms subserving ATP action in gonadotropes. Addition of ATP produced a biphasic intracellular Ca2+ (Ca2+i) response: a transient spike followed by a small plateau. Removal of extracellular Ca2+ or depolarization with KCl abolished the plateau but had no effect on the spike. The plateau was also blocked by cadmium or nifedipine but not nickel. Pretreatment with GnRH or thapsigargin but not ryanodine inhibited the subsequent Ca2+i response to ATP. Pertussis toxin had no effect on ATP-induced Ca2+i response, whereas the phospholipase C inhibitor U73122 reduced the response. These observations suggest that the Ca2+i response is mediated by a pertussis toxin-insensitive and phospholipase C-coupled G-protein and reflects Ca2+ release from the GnRH- and thapsigargin-sensitive Ca2+ pool followed by Ca2+ influx through high voltage-gated Ca2+ channels. Activation of these ATP receptors had no apparent effects on the cAMP and cGMP signaling systems. Treatment with ATP-gamma S caused the translocation of protein kinase C (PKC) epsilon but not PKC zeta and PKC alpha to the particulate fraction. These data not only characterize the ATP receptor-mediated intracellular signaling in alpha T3-1 cells and render further evidence for a mediator role for nucleotides in gonadotrope function but also provide the first direct demonstration of PKC translocation by ATP receptors.

GH synthesis and release from the anterior pituitary is governed by the opposing actions of somatostatin (SS) and GH-releasing factor (GRF), derived from the periventricular and arcuate nucleus (ARC) of the hypothalamus respectively. GH is known to regulate its own release by hypothalamic autofeedback mechanisms, but the extent to which this is a direct effect rather than indirectly via the generation of IGFs is still a subject of debate. GH receptors are known to be present in the hypothalamus, but their physiological regulation is poorly understood. We therefore used in situ hybridization histochemistry to investigate the effects of GH status on hypothalamic GH receptor gene expression, using hypophysectomized normal and dw/dw dwarf rats as models of acquired and congenital GH deficiency. Hypophysectomy resulted in a timedependent reduction in GH receptor gene expression. ARC GH receptor transcripts in untreated dw/dw dwarf rats were half those found in normal animals of the same background strain (16·8±1·7 vs 9·3± 1·9 d.p.m./mg, Pdw/dw rats to normal. In contrast, central infusions of hGH at 26·4 and 79·2 μg/day for 6 days in normal rats lowered ARC GH receptor gene expression. The sensitivity of GH receptor gene expression within the central nervous system to peripheral and central GH levels suggests that feedback regulation of GRF and/or SS may be mediated directly by these receptors, and that the sensitivity to GH feedback is also subject to autoregulation by GH altering its own receptor expression. Journal of Endocrinology (1995) 147, 225–234

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of external ATP on [Ca2+]i in cultured rat hypothalamic neurons. The addition of ATP at microM concentrations, but not adenosine, AMP, ADP or GTP, produced a rapid, dose-dependent increase in cytosolic Ca2+. The hydrolysis-resistant ATP analogues 3-thio-ATP and beta,gamma-imido-ATP produced a similar response but alpha,beta-methylene ATP had much lower efficacy. The ATP response was inhibited by 10 microM nifedipine, abolished by 50 microM cadmium and by the absence of extracellular Ca2+, but was unaffected by ryanodine or omega-conotoxin GVIA. The P2-purinoceptor antagonist suramin reversibly and selectively inhibited the ATP response but had no effect on other neurotransmitter-induced Cai2+ responses. Antagonists to muscarinic, nicotinic, NMDA, non-NMDA, GABA, 5-HT and adenosine receptors had no effect on the ATP response. Thus the Ca2+ response of hypothalamic neurons to ATP is mediated by specific suramin-sensitive ATP-receptors, activation of which is independent of ATP hydrolysis and results in an influx of extracellular Ca2+ largely through high voltage-gated Ca2+ channels. These findings support the assertion that ATP acts in the CNS as an excitatory neurotransmitter.

We have used in situ hybridization histochemistry to investigate interactions between the thyroid and adrenal axes in the rat. Propylthiouracil (PTU)-induced hypothyroidism caused a significant reduction in CRH gene transcripts in the paraventricular nucleus of male rats, with a concomitant decrease in both POMC gene expression in the anterior pituitary gland and circulating corticosterone. Conversely, adrenalectomy alone or adrenalectomy with high dose corticosterone pellet replacement had no significant effect on the thyroid hormone axis. The effect of thyroid hormones on the adrenal axis was not secondary to changes in the level of circulating corticosteroids, as the same effect of PTU-induced hypothyroidism was seen in adrenalectomized rats given corticosterone replacement. Neither was the effect of PTU simply the result of a direct toxic effect on the hypothalamus, as concurrent treatment with T3 blocked the response. We conclude that circulating levels of thyroid hormones have a major effect on the central regulation of the hypothalamic-pituitary-adrenal axis.

Pit-1, a member of the POU family of homeodomain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein—DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes. The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.

The pituitary gland influences osmoregulation, growth, lactation, the immune system and parturition, and is respon- sible for regulating thyroid, adrenal and gonadal function. During lactation and in response to conditions such as hypothyroidism and hypoadrenalism, specific cell types in the pituitary undergo hypertrophy. For reasons that are as yet unclear, tumour formation, albeit subclinical, is remark- ably common. Approximately 10% of all clinically overt intracranial neoplasms arise from the pituitary gland and at autopsy, if histological examination of unselected autopsy material is to be believed, adenomas are present in as many as one in five (Costello, 1936; Kontogeorgos

In the rat, 48 h of food deprivation significantly reduces hypothalamic paraventricular nucleus (PVN) thyrotropin-releasing hormone (TRH) gene expression, anterior pituitary thyrotropin (TSH) gene expression and circulating triiodothyronine (T3). Using in situ hybridization histochemistry, we have now assessed the effect of selective nutritional deprivation, by comparing protein-free and protein and fat-free diets with a normal diet matched for total energy content. As previously demonstrated, fasting markedly reduced PVN TRH transcripts, pituitary TSB beta transcripts, circulating T3 and body weight. Compared to rats fed a control diet, rats fed a protein-free or a protein and fat-free diet of similar energy content showed a highly significant decrease in PVN TRH transcripts, pituitary TSB beta transcripts and circulating T3 levels. The exclusion of fat from the protein-free diet did not produce any further decline in the parameters measured. This indicates that variations in the protein composition alone of the diet are sufficient to reduce hypothalamic TRH mRNA, pituitary TSB beta mRNA and plasma T3, and are the predominant factors in the TRH response to starvation.

We have used in situ hybridization histochemistry to determine the effect of hippocampal-hypothalamic disconnection on hypothalamic thyrotrophin-releasing hormone (TRH) and anterior pituitary thyrotrophin beta subunit (TSH beta) transcripts in adult male CFY rats. Electrothermal lesions of the fornix pathway significantly increased TRH and TSH transcripts and increased circulating levels of triiodothyronine (T3). Fornix transection did not, however, prevent feedback regulation of TRH and TSH transcripts during exogenous T3-induced hyperthyroidism or propylthiouracil-induced hypothyroidism. Hippocampal inputs to the hypothalamus contribute to setting the basal activity of the thyroid axis, but do not mediate the feedback effects of T3.

In rats, a shift from somatotroph dominance to lactotroph dominance during pregnancy and lactation is well reported. Somatotroph to lactotroph transdifferentiation and increased lactotroph mitotic activity are believed to account for this and associated pituitary hypertrophy. A combination of cell death and transdifferentiation away from the lactotroph phenotype has been reported to restore non-pregnant pituitary proportions after weaning. To attempt to confirm that a similar process occurs in mice, we generated and used a transgenic reporter mouse model (prolactin (PRL)-Cre/ROSA26-expression of yellow fluorescent protein (EYFP)) in which PRL promoter activity at any time resulted in permanent, stable, and highly specific EYFP. Triple immunochemistry for GH, PRL, and EYFP was used to quantify EYFP+ve, PRL−ve, and GH+ve cell populations during pregnancy and lactation, and for up to 3 weeks after weaning, and concurrent changes in cell size were estimated. At all stages, the EYFP reporter was expressed in 80% of the lactotrophs, but in fewer than 1% of other pituitary cell types, indicating that transdifferentiation from those lactotrophs where reporter expression was activated is extremely rare. Contrary to expectations, no increase in the lactotroph/somatotroph ratio was seen during pregnancy and lactation, whether assessed by immunochemistry for the reporter or PRL: findings confirmed by PRL immunochemistry in non-transgenic mice. Mammosomatotrophs were rarely encountered at the age group studied. Individual EYFP+ve cell volumes increased significantly by mid-lactation compared with virgin animals. This, in combination with a modest and non-cell type-specific estrogen-induced increase in mitotic activity, could account for pregnancy-induced changes in overall pituitary size.

An 18-year-old woman presented to us in June, 2004, at 27 weeks’ gestation with aproteinuric hypertension (178/111 mm Hg) and intermittent panic attacks (palpitations, left abdominal pain, and perspiration). 24-h urinary catecholamine excretion was >20 000 nmol. Ultrasonography showed a 6·9 cm × 3·6 cm × 4·6 cm structure obscuring the left adrenal gland. Haemodynamic stability was achieved with phenoxybenzamine and labetolol, for the presumptive diagnosis of phaeochromocytoma. A staging CT was undertaken before elective caesarean at 37 weeks’; a healthy infant was delivered. The tumour was resected a month later. Histologically the tumour arose from the sympathetic paraganglion cells; the left adrenal gland itself was normal. Some vascular invasion was noted. DNA analysis showed VHL and RET genes to be normal, but there was a succinate dehydrogenase complex subunit B (SDH-B) gene mutation, predisposing her to paraganglioma syndrome type I. Postoperative ultrasound showed a 3·2 cm mass anterior to the inferior vena cava at the aortic bifurcation, but the patient declined further investigation. She remained asymptomatic until the 32nd week of her next pregnancy, when fl ushing and palpitations were associated with rising urinary catecholamine secretions (to 4423 nmol/day). MRI showed that the mass had grown slightly to 3·5 cm. Despite an asystolic episode during caesarean section at term, the patient survived to undergo laparoscopic resection of the tumour 6 weeks’ postpartum. Again, paraganglioma was identifi ed histologically. The patient remained asymptomatic at follow-up in April, 2009. Paragangliomas account for only 10–15% of catecholamine-secreting tumours. A large series put the prevalence of paraganglioma and phaeo chromocytoma during pregnancy at fi ve per million. Maternal and fetal mortality are 4% and 11%, respectively, which is related to delay in presentation and diagnosis. In pregnancy phaeochromocytomas may initially be missed, because the symptoms can mimic pre-eclampsia (table). Urinary catecholamine secretion and plasma metanephrines are not raised in normal pregnancy or pre-eclampsia and confi rm the diagnosis of phaeochromo cytoma. Adequate medical management is required before delivery and surgery. Inpatient observation and management are recommended because of the unpredictable nature of phaeochromocytoma in pregnancy, although successful outpatient management was achieved when our patient declined admission. Resection should promptly follow medical stabilisation in cases diagnosed prior to 24 weeks’; after this time maintenance medical therapy should continue to optimise fetal maturity. Elective caesarean allows close monitoring and is the preferred mode of delivery since labour can be lethal. Planned caesarean also off ers the opportunity for simultaneous tumour resection, but delaying this into the puerperium decreases vascularity and improves access. Germline mutations account for a quarter of phaeochromocytomas; commonly disease is of young onset and is multifocal or extra-adrenal. Associated familial syndromes including Von Recklinghausen’s disease, Von Hippel-Lindau disease, and multiple endocrine neoplasia types IIa and IIb. Mutations in SDH-B, SDH-C, and SDH-D genes account for 33% of patients with phaeochromocytoma due to genetic abnormalities. SDH genes code for discrete subunits of the mitochondrial enzyme complex II (involved in Kreb’s cycle) and act as classic tumour suppression genes. SDHB mutations are associated with a 33% risk of recurrence and malignancy, and SDH-D mutations with an increased risk of multifocal paragangliomas, and renal and thyroid carcinomas. In women of reproductive age, genetic testing for one of the familial syndromes associated with phaeochromocytoma should be considered when there is family history of an associated syndrome, if the patient has any clinical features associated with a syndrome, or if the woman has extra-adrenal or multifocal disease. Although, the penetrance of RET and VHL is understood, the proportion of women with SDH mutations who develop clinical disease is unknown, complicating genetic counselling. Women known to have syndromes associated with phaeochromocytomas should receive preconceptual counselling about risks of disease developing during pregnancy.

We have described a patient with a thyrotrophin-secreting pituitary adenoma and correlated a detailed physiological and anatomical investigation of the surgically resected tumour with its in vivo regulation. Thyrotrophin secretion was inhibited by circulating thyroid hormones, dopaminergic agonists and the somatostatin analogue SMS 201-995 but could not be stimulated by thyrotrophin-releasing hormone or further inhibited by exogenous triiodothyronine. Prolonged treatment with SMS 201-995 caused tumour shrinkage as shown by successive computed tomography scans but was accompanied by tumour desensitization and the development of diabetes mellitus. This is the first thyrotroph adenoma in which somatostatin receptors have been directly demonstrated and shown to completely block thyrotrophin-releasing hormone-induced inositol phospholipid accumulation when occupied. In addition, preincubation with triiodothyronine significantly inhibited thyrotrophin-releasing hormone-induced inositol phospholipid turnover in dispersed pituitary cells indicating that in this tumour, circulating thyroid hormones were exerting feedback inhibition at the level of the pituitary either by reducing the number of thyrotrophin-releasing hormone receptors and/or their coupling to second messenger pathways. In keeping with this hypothesis, the acute reduction in intrapituitary triiodothyronine by sodium ipodate in vivo had no effect on peripheral thyrotrophin over 6 h suggesting that the onset of the effect of triiodothyronine withdrawal on thyrotrophin secretion was suitably delayed. The importance of the inositol phospholipid second messenger pathway in transducing the secretory response in this tumour was further corroborated by electrophysiological studies which demonstrated thyrotrophin-releasing hormone-induced changes in K(+) currents which are dependent on intracellular Ca(2+) ions, presumably mobilized via the inositol phospholipids. In addition to thyrotrophin and alpha subunit, growth hormone mRNA and growth hormone were found throughout the tumour as were two populations of cells distinguished electron microscopically by the size of their secretory granules. Although acromegalic features are not unusual in thyrotroph adenomas, our patient showed no evidence of inappropriate growth hormone secretion during surgery or in response to pre- or post-operative insulin stress tests.

SUMMARY We have studied the inositol phospholipid turnover response to thyrotrophin-releasing hormone (TRH) gonadotrophin-releasing hormone (GnRH) and arginine vasopressin (AVP) in five corticotroph and six somatotroph pituitary adenomas. GnRH (100 nM) increased inositol phospholipid turnover in five of five somatotroph adenomas tested by an average of 36.4 |Mp 9.4% (mean |Mp SE). In a fifth, which was too small to allow inositol phospholipid turnover to be assessed, GnRH produced a rapid increase in mean intracellular calcium ion concentration. Only one of five corticotroph adenomas responded to GnRH with an increase in inositol phospholipid turnover of 19%. In contrast, all the somatotroph and corticotroph adenomas tested (four of four and five of five respectively) increased inositol phospholipid turnover in response to AVP (100 nM), by an average of 61 |Mp 11.8% and 415 |Mp 186% respectively. The finding of an inositol phospholipid or intracellular Ca2+ response to thyrotrophin-releasing hormone (TRH) (100 nM) in three of five somatotroph adenomas (Ca2+ increasing from 50 to 175 nM in one and inositol phospholipid turnover increasing by 172 |Mp 1.9% and 49 |Mp 5.2% in two) and to GnRH in all five somatotroph adenomas concurs with the common clinical finding of paradoxi-cal growth hormone responses to these releasing factors in patients with acromegaly. The lack of such a response in three of the four corticotroph adenomas suggests that the appearance of GnRH and TRH receptors on adenomatous pituitary cells is not common to all cells.

Vasopressin ribonucleic acid (VP RNA) accumulates in the posterior lobe of the rat pituitary in response to an osmotic stimulus. The accumulation of posterior pituitary VP RNA can be prevented by stalk transection or the intracerebroventricular injection of colchicine. The hypothalamic and pituitary VP RNAs however, have different structures and are independently regulated. Depletion of serotonin with parachlorophenylalanine blocks the osmotically induced accumulation of both VP and oxytocin RNA in the hypothalamus but does not affect the accumulation of VP RNA in the posterior pituitary gland. In Brattleboro rats, posterior lobe oxytocin RNA but not VP RNA, increases after osmotic stimulation. Although axonal transport of RNA may occur, our results are also consistent with VP RNA synthesis in pituicytes controlled by hypothalamic neuroendocrine factors released by neurohypophyseal terminals in response to osmotic stimuli.

The novel somatostatin receptor agonist pasireotide binds with high affinity to somatostatin receptors SSTR1, 2, 3, and 5. Acting principally through the latter, it inhibits basal and CRH-stimulated ACTH secretion from the AtT20 corticotroph cell line and ACTH release from a proportion of human corticotroph adenomas both in vitro and in vivo. Data supporting an additional antiproliferative effect has led to pasireotide being explored as a potential therapy for patients with Cushing's disease. We have compared the effects of pasireotide and octreotide on adrenalectomy-induced mitotic and apoptotic activity in the male rat anterior pituitary. Adrenalectomized rats were treated with daily sc injections of vehicle, pasireotide, or octreotide. Changes in proliferation and apoptosis were determined 2-6 d postoperatively. Pasireotide and octreotide had no effect on baseline pituitary cell turnover and no measurable effects on apoptosis. However, the wave of increased mitotic activity normally seen in the pituitary after adrenalectomy was completely abolished. Nevertheless, pasireotide and octreotide did not diminish the increase in ACTH-immunopositive cell index after adrenalectomy, indicating that cell division and differentiation of hormonally null cells in the pituitary are under independent control. In conclusion, basal cell turnover in the pituitary is not inhibited by pasireotide or octreotide. Bilateral adrenalectomy stimulates differentiation of preexisting null cells into ACTH-positive cells. Cell division after bilateral adrenalectomy occurs in a specific subpopulation of hormonally null cells that are equally sensitive to the antiproliferative effects of pasireotide and octreotide, implicating SSTR2 receptors in this antimitotic response.

OBJECTIVES: Glucocorticoid withdrawal in man is associated with transient but sometimes prolonged impairment of hypothalamo-pituitary-adrenal axis secretory responsiveness. This has led to continued concern in the clinical arena. The acute anterior pituitary response to glucocorticoids in the rat includes apoptosis-mediated deletion of a cell population. Whilst continued cell turnover following glucocorticoid withdrawal and the potential for differentiation of uncommitted precursor cells and transdifferentiation of other secretory cell types predicts that, given sufficient time, complete anterior pituitary trophic recovery is likely, this hypothesis has not previously been tested. DESIGN AND METHODS: We have quantified pituitary mitotic and apoptotic rate, as well as corticotroph number and pro-opiomelanocortin transcript prevalence, together with hypothalamic corticotrophin-releasing hormone and vasopressin transcript prevalence 5 weeks after three short dexamethasone treatments each separated by a week. Bilateral adrenalectomy was then carried out as a maximal secretory and trophic stimulus, and the response to a fourth dexamethasone treatment assessed 1 week later. RESULTS: Anterior pituitary mitotic index was significantly higher in rats previously exposed to dexamethasone compared with age-matched controls exposed to dexamethasone for the first time. No differences were found in the subsequent apoptotic response to a fourth dexamethasone treatment or in the levels of paraventricular corticotrophin-releasing hormone or vasopressin transcripts or pituitary pro-opiomelanocortin transcripts. CONCLUSIONS: These data indicate that full recovery of pituitary mitotic activity takes longer than the recovery of secretory parameters, and suggest that, for several weeks after glucocorticoid exposure, the ability of the pituitary to meet fresh demands placed on it may be suboptimal.

Depending on timing and dose, exogenous glucocorticoids induce a wave of apoptosis in the adult rat anterior pituitary, a response that is enhanced by adrenalectomy. In this study, we show that the size of the glucocorticoid-sensitive apoptotic population progressively increases during the week following surgical adrenalectomy, plateaus for a further week, then spontaneously declines to levels seen in intact animals by 4 wk. Mitotic activity, in contrast, rises rapidly post adrenalectomy but returns to baseline within 2 wk. Increased mitotic activity precedes the increase in the population of cells that undergo glucocorticoid-induced apoptosis and the subsequent decline in mitotic activity precedes the decline in apoptotic sensitivity despite persistent elevation of hypothalamic CRH and pituitary proopiomelanocortin transcripts. If glucocorticoid exposure is delayed until 4 wk post adrenalectomy when the apoptotic response has returned to baseline, glucocorticoid withdrawal, by transiently increasing mitotic activity, again primes the formation of an expanded glucocorticoid-sensitive apoptotic cell population. These data suggest that apoptotic sensitivity is largely confined to cells that have recently entered the cell cycle. This observation is further corroborated by demonstrating an abrupt glucocorticoid-induced step-down in the bromodeoxyuridine-labeling index to basal levels in rats given daily injections of bromodeoxyuridine during the week following adrenalectomy.

A complete inventory of pituitary trophic responses depends on precise estimates of mitotic activity and apoptotic events, and accurate characterization and quantification of pituitary cell subtypes irrespective of previous and current physiological demand. For a discrete structure that has been so extensively studied, it is disappointing but perhaps not surprising that none of these measures is available and therefore that the relative contributions to changes in the proportions of pituitary cellular subpopulations of trophic activity, differentiation of pluripotent cells and variations in the secretory profiles of apparently committed cells remain almost impossible to determine. To fully appreciate the extent of this dilemma, it should be remembered that conservative estimates of the proportion of corticotrophs in the rat anterior pituitary under basal conditions vary over twofold and that it is still not clear whether the apparent threefold increase in mammotrophs during pregnancy is the result of maturation of uncommitted cells, transdifferentiation of other cells such as somatotrophs, cell division, or a mixture of all three. Equally, while it has been known for some time that adrenalectomy results in a transient increase in anterior pituitary mitotic activity and appropriately timed supraphysiological glucocorticoid replacement with a wave of apoptosis, the precise identity of the cells involved in both of these responses is open to question. Thus, although many of the physiological stimuli associated with apparent changes in the proportions of pituitary cellular subpopulations are known, the precise mechanism of the changes and the consequences of the same remain obscure. This review summarizes the limited literature on pituitary trophic activity and asks what, if anything, analysis of pituitary trophic activity using current technology can tell us.